Neuroligins (NLGNs) are brain-specific cell adhesion molecules. They are expressed on the postsynaptic membrane and bind to presynaptic neurexins (NRXNs) spanning the synaptic cleft. Interestingly, mutations in both NLGNs and NRXNs have been identified in Autism Spectrum Disorder (ASD) patients. This has led researchers to develop genetically engineered NLGN mouse models to study the etiology of ASDs. These studies have shown that NLGN dysfunction can shift the balance of inhibition and excitation in the brain. NLGN isoforms are highly conserved, yet display distinct synaptic localizations. However, the molecular mechanisms that regulate isoform-specific targeting and localization are not well understood. We focus on the role of protein-protein interactions and post-translational modifications in dictating NLGN trafficking and functional regulation. Over the last few years we have identified several different phosphorylation sites on the different neuroligin isoforms. We have been characterizing the kinases involved and the physiological relevance to synapse formation. ASDs are a group of neurodevelopmental disorders that have a high genetic predisposition and higher occurrence rates in males than females. A variety of point mutations have been identified in X-linked NLGN3 and 4X in patients with intellectual disability and symptoms characteristic of ASDs. Interestingly, all of the autism-associated point mutations in NLGN3 and NLGN4X reported thus far reside in their extracellular domains except for a single point mutation in the intracellular domain of NLGN4X at arginine (R) 704, which is modified to a cysteine (C). We discovered that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707 in human embryonic neurons. This autism mutation (R704C) eliminates T707 phosphorylation, which is critical for NLGN4X-mediated excitatory enhancement. Interestingly, unlike other NLGN ASD-associated mutations, R704C, did not disrupt the stability or surface expression of NLGN4X, yet still led to synaptic dysfunction. Mouse models of autism have uncovered a role for an imbalance of excitatory/inhibitory transmission, often resulting in direct increases in inhibitory transmission. Our results establish a potential causality between a genetic mutation, a key posttranslational modification, and robust synaptic changes and will provide insights in elucidating the pathophysiology of ASDs. In human there is also NLGN-4Y, which is located on the Y chromosome and is almost identical to NLGN-4X. In fact, NL-4X and NL-4Y have only eight amino acid differences in the extracellular domain and five in the intracellular domain. However, there are no studies on phosphorylation of NLGN-4Y. We have now compared the PKC phosphorylation of 4X vs 4Y using different kinases in conjunction with mass spectrometry, and find that NL-4X and NL-4Y are phosphorylated at different residues. Importantly, there is a difference in the levels of the phosphorylation of the PKC T707 site. These results suggest that NL-4X and NL-4Y are regulated in a fundamentally different manner, and we are following up on these findings. We believe through a better investigation of the sex-linked isoforms of NLGNs, we hope to understand the sex bias associated with ASDs. NLGN-1 and the scaffolding protein PSD-95 are both localized at excitatory synapses. In addition, NLGN-1 binds to PSD-95 through the PDZ ligand in the cytoplasmic tail. We have identified a protein kinase A (PKA) phosphorylation site on NLGN-1, near the PDZ ligand, in vitro and in heterologous cells. When we introduce a phospho-mimetic mutation at the NLGN-1 PKA site, the interaction between NLGN-1 and PSD-95 is decreased in vitro and in situ. Therefore, we find that phosphorylation regulates PSD-95 binding to NLGN-1, just as we have shown for NMDARs. We also have demonstrated that phosphorylation regulates NLGN-2, the neuroligin isoform that is located and specifically functions at inhibitory synapses. In particular, NLGN-2 is a substrate for PKA as detected using mass spectrometry. We have generated a phospho-specific antibody against this site, and demonstrate its specificity. With this great tool, we are now characterizing NLGN-2 phosphorylation and regulation in vivo. NLGN-2 phosphorylation is regulated by synaptic activity, and we are investigating the precise mechanisms by which this occurs. Finally, neuroligins undergo cleavage of their extracellular domain in response to synaptic activity. We have uncovered an isoform-specific regulation of this cleavage and are studying the mechanisms underlying its regulation. In particular, we are studying the proteases involved in neuroligin cleavage and the molecular determinants within the neuroligins.